Detecting the phosphate status of phytoplankton by enzyme-labelled fluorescence and flow cytometry.

The novel phosphatase substrate, ELF-97 phosphate, yields intensely green fluorescent precipitates of ELF-97 alcohol (ELFA) upon enzymatic dephosphorylation, and thereby traces phosphatase activity back to its producer. In this study, we show that ELFA fluorescence is a useful tool in flow cytometric analysis of natural phytoplankton populations. Presence of endogenous fluorescent pigments allowed flow cytometric distinction of clusters in the phytoplankton community in Lake Loosdrecht (The Netherlands): Eukaryotes (diatoms and green algae), chlorophyll a and b containing but phycobilin-less cyanobacteria (Prochlorothrix hollandica), and phycocyanin-containing cyanobacteria (predominantly Limnothrix sp.). Several, but not all tested cyanobacteria showed ELFA fluorescence. The dominant Limnothrix sp. possesses a derepressible phosphatase, whereas the second most abundant strain, P. hollandica, did not have phosphatase activity. Within both natural and cultured populations of Limnothrix sp. we found discernible levels of ELFA fluorescence, indicating the presence of subpopulations with different physiological characteristics.